Turhan Markussen

Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity

In this study we characterize two proteins encoded by the two smallest genomic segments of the piscine orthomyxovirus infectious salmon anaemia virus (ISAV). Both proteins, encoded by the un-spliced ORF from genomic segment 7 (s7ORF1) and the larger ORF from segment 8 (s8ORF2), are involved in modulation of the type I interferon (IFN) response. The data suggests that the s7ORF1 protein is collinearly encoded, non-structural, contains no nuclear localisation signals, localises mainly to the cytoplasmic perinuclear area and does not bind single- or double-stranded RNA. On the other hand, genomic segment 8 uses a bicistronic coding strategy and the encoded s8ORF2 protein is a structural component of the viral particle. This protein contains two nuclear localisation signals, has a predominantly nuclear localisation, binds both double-stranded RNA and poly-A tailed single-stranded RNA, but not double-stranded DNA. In poly I:C stimulated salmon cells both ISAV proteins independently down-regulate the type I IFN promoter activity. Thus, ISAV counteracts the type I IFN response by the action of at least two of its gene products, rather than just one, as appears to be the case for other known members of the Orthomyxoviridae.

Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R267 in the fusion (F) protein, suggesting a Q266-->L266 substitution to be a prerequisite for virulence. To gain virulence in isolates lacking this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus.

Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Use of molecular epidemiology to trace transmission pathways for infectious salmon anaemia virus (ISAV) in Norwegian salmon farming.

BACKGROUND Infectious Salmon Anaemia (ISA) is a disease affecting farmed Atlantic salmon, and most salmon producing countries have experienced ISA outbreaks. The aim of the present study was to use epidemiological and viral sequence information to trace transmission pathways for ISA virus (ISAV) in Norwegian salmon farming. METHODS The study covers a period from January 2007 to July 2009 with a relatively high rate of ISA outbreaks, including a large cluster of outbreaks that emerged in Northern Norway (the North-cluster). Farms with ISA outbreaks and neighbouring salmon farms (At-risk-sites) were tested for the presence of ISAV, and epidemiological information was collected. ISAV hemagglutinin-esterase (HE) and fusion (F) protein genes were sequenced and phylogenetic analyses were performed. Associations between sequence similarities and salmon population data were analysed to substantiate possible transmission pathways. RESULTS There was a high degree of genetic similarity between ISAV isolates within the North-cluster. ISAV was detected in 12 of 28 At-risk-sites, and a high proportion of the viruses were identified as putative low virulent genotypes harbouring the full length highly polymorphic region (HPR); HPR0 of the HE protein and the amino acid glutamine (Q) in the F protein at position 266. The sequences from HPR0/F (Q(266)) genotypes revealed larger genetic variation, lower viral loads and lower prevalence of infection than HPR-deleted genotypes. Seaway distance between salmon farms was the only robust explanatory variable to explain genetic similarity between ISAV isolates. DISCUSSION We suggest that a single HPR-deleted genotype of ISAV has spread between salmon farms in the North-cluster. Furthermore, we find that HPR0/F (Q(266)) genotypes are frequently present in farmed populations of Atlantic salmon. From this, we anticipate a population dynamics of ISAV portrayed by low virulent genotypes occasionally transitioning into virulent genotypes, causing solitary outbreaks or local epidemics through local transmission.

Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon

Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (Salmo salar) farming. HSMI is associated with Piscine orthoreovirus (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus

Abstract Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser32 to Ala32 amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser32 amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzymes catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure–function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

The non-structural protein μNS of piscine orthoreovirus (PRV) forms viral factory-like structures

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation in farmed Atlantic salmon. The virus is ubiquitous and found in both farmed and wild salmonid fish. It belongs to the family Reoviridae, closely related to the genus Orthoreovirus. The PRV genome comprises ten double-stranded RNA segments encoding at least eight structural and two non-structural proteins. Erythrocytes are the major target cells for PRV. Infected erythrocytes contain globular inclusions resembling viral factories; the putative site of viral replication. For the mammalian reovirus (MRV), the non-structural protein μNS is the primary organizer in factory formation. The analogous PRV protein was the focus of the present study. The subcellular location of PRV μNS and its co-localization with the PRV σNS, µ2 and λ1 proteins was investigated. We demonstrated that PRV μNS forms dense globular cytoplasmic inclusions in transfected fish cells, resembling the viral factories of MRV. In co-transfection experiments with μNS, the σNS, μ2 and λ1 proteins were recruited to the globular structures. The ability of μNS to recruit other PRV proteins into globular inclusions indicates that it is the main viral protein involved in viral factory formation and pivotal in early steps of viral assembly.

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

Low virulent infectious salmon anaemia virus (ISAV) replicates and initiates the immune response earlier than a highly virulent virus in Atlantic salmon gills

Observations from the field and experimental evidence suggest that different strains of infectious salmon anaemia virus (ISAV) can induce disease of varying severity in Atlantic salmon. Variation in host mortality and dissemination of ISAV isolates with high and low virulence was investigated using immersion challenge; from which mortality, pathological, immunohistochemical and preliminary molecular results have been previously published. Here, real-time RT-PCR analysis and statistical modelling have been used to further investigate variation in virus load and the response of four select immune genes. Expression of type I and II interferon (IFN), Mx and γIFN induced protein (γIP) to high and low pathogenic virus infection were examined in gill, heart and anterior kidney. In addition, a novel RNA species-specific assay targeting individual RNA types was used to investigate the separate viral processes of transcription and replication. Unexpectedly, the low virulent ISAV (LVI) replicated and transcribed more rapidly in the gills compared to the highly virulent virus (HVI). Subsequently LVI was able to disseminate to the internal organs more quickly and induced a more rapid systemic immune response in the host that may have offered some protection. Contrary to this, HVI initially progressed more slowly in the gills resulting in a slower generalised infection. However HVI ultimately reached a higher viral load and induced a greater mortality.

Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.